Title Purification and Cellular Localization of Extracellular Nuclease of Serratia marcescens Expressed in Escherichia coli
Author Kim, Woe Yeon¹'³ · Lee, Hoon Sil¹'³ · Suh, Sook Jae² · Cho, Moo Je¹'³ · Lee, Sang Yeol¹'³ · Kim, Jae Won *
Address ¹Department of Biochemistry; ²Department of Biology; Department of Microbiology; ³Plant Molecular Biology and Biotechnology Research Center(PMBBRC), Gyeongsang National University
Bibliography Korean Journal of Microbiology, 32(2),147-154, 1994
Key Words Serratia marcescens, nuclease, secretion, localization
Abstract Nuclease was secreted to the environmental media from the Escherichia coli JM107 tranformant harboring the extracellular nuclease gene of Serratia marcescens in the plasmid of pNUC4. Under the growth conditions, the amount of secreted enzyme was increased in parallel with bacterial growth conditions, the amount of secreted enzyme was increased in parallel with bacterial growth. The enzyme was purified using chromatofraphic procedures of Matrex green gel and heparin agarose affinity gel, resulted in 50-fold purification with 15% recovery of the enzyme. The apparent molecular weight of the enzyme was estimated to be 29Kda by sodium dodecylsulfate denaturing gel electrophoresis. Using the purified enzyme, polyclonal antibody was obtained from the rabbit. The specificity of the antibody was confirmed by immunoblotting and immunoprecipitaion. For the investigation of cellular distribution of the enzyme, cells were fractionated into three fractions; cytoplasm, periplasm and extracellular fluid. While more than 80% of the enzymatic activity was detected in the extracellular fluid and periplasm, a little was found in the cytoplasm, indicating that the enzyme was likely to be immediately exported to the membrane for excretion after biosynthesis. These results were confirmed again by immunocytochemistry technique using the antibody.
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